Membrane Attack Complexes Associated with Circulating Immune Complexes

ABSTRACT

The invention relates to demonstrate the presence of non covalently linked complement proteins with CIC. Invention demonstrate the presence of C5 and MAC on CIC. The invention provides methods to measure M complement proteins C1q, C3, C4, C5 bound to CIC as markers of disease activity and pathogenicity for complement and CIC mediated diseases. The invention also relates to modifications and blocking of formation of MAC on the CIC by developing a useful reagent that can be monoclonal antibody, active molecule, mimotope or peptide molecule. The invention describes usefulness to reduce the non-covalent complement components, formation of MAC and association of C5 to CIC for therapeutic application in pathological disease with involvement of CIC and complement.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. application 60/______ in,filed Jan. 28, 2004, which is fully incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

REFERENCE TO A SEQUENCE LISTING

Not applicable.

BACKGROUND OF THE INVENTION

Complement pathway is made of more than 35 plasma proteins that play akey role in host defense against microbial infections and continuousclearance of apoptotic debris. The formation of an antigen-antibodycomplex (also referred to as immune complex) is the principal mechanismof complement activation. Complement plays a key role in the antigenpresentation and regulation of antibody responses by B cells. Theactivation of complement proteins primarily occurs by three majorpathways—classical, alternate and mannose binding lectin pathways. Eachpathway leads to formation of C3 convertase which then cleaves the C3complement protein to generate C3a, an anaphylotoxin and a secondsubunit, C3b, that participates in formation of C5 convertase. At the C5convertase level the three complement pathways converge leading tocleavage of the C5 molecule that results in formation of C5b and C5a,another potent anaphylotoxin. The C5b complement protein forms thesubstrate for formation of a macromolecular complex by associating withthe complement proteins C6, C7, C8, and up to sixteen molecules of C9,to form the terminal complement complex (TCC), also called a membraneattack complex (MAC), or simply, C5b-9. The MAC formation occurs oncethe complement protein C5 is cleaved into C5a and C5b. It is known thatonce the C5 convertase splits C5 into C5a and C5b, C5b then associateswith C6 and C7 to form C5b-7, which complex then inserts into the cellmembrane. Once the C5b-7 becomes inserted into the cell wall, it thenacts as substrate for addition of the remaining components of MACcomplex namely C8 and C9 on the cell membrane. The activation ofcomplement pathway by CIC (circulating immune complexes) subsequentlyleads to the formation of a high number of MAC molecules on CIC. TheseCICs transfers high doses of MAC to cell membrane thus inflictingreversible cell damage leading to cell lysis. The amount of MACtransferred to cell membrane determines whether the cell will bedestined for apoptosis or necrosis. Sublytic doses of MAC on cell wallstriggers multiple signaling pathways initiating pleiotropic responses.

Complement plays a key role in regulating the innate and adoptive immuneresponses. The activation of complement pathway and subsequentgeneration of C5a and MAC (TCC, C5b-9) by polymerization of terminalcomponents of the complement are the key mediators of complement inducedsignaling pathways and inflammatory responses. In the present invention,for the first time applicant demonstrates the presence of C5 and MACcomplexes on the CIC and demonstrates that the CIC act as the substratefor the formation of MAC and their subsequent transfer to the cellmembrane. Applicant also shows that the complement activation productsC1q, C3, C4, C5 and C5b-9 present on CIC are non-covalently linked toCIC. Applicant also demonstrates that CIC carrying higher levels ofcomplement are present in the serum of patients during disease activity,e.g., for rheumatoid arthritis, systemic lupus erythematosus, Chemokineproduction by alveolar macrophages in the presence of C5a or C5b-9[Czermark et al, Am. J. Pathol. 154(5):1513 (1999)] is significantlyhigher after treatment with circulating immune complexes (CIC) composedwith IgG isotype of immunoglobulin. It was noted that for production ofmacrophage inflammatory protein-2 (MIP-2), cytokine-induced neutrophilchemoattractant (CINC), monocyte chemoattractant protein-1 (MCP-1) andmacrophage inflammatory protein-1α (MIP-1 α). by C5a and or C5b-9 in anintrapulmonary environment, the presence of IgG containing CIC wasessential. [Czermak et al, Am. J. Pathol. 154(5):1513 (1999)] The dataaccumulated over years have supported the notion that the formation ofC5 convertase leading to generation of C5a and C5b-9 is a majorcomponent of chronic inflammatory processes associated withatherosclerosis [Yasojima et al, Am. J. Pathol. 158(3):1039 (2001)],myocardial infarction and myocardial ischemia and reperfusion injury[Vaveka et al, Circulation 97:2259 (1998); Afanasyeva et al, Am. J.Pathol. 161(2):351 (2002)].

In view of the significance of the activation of the C5 molecule andsubsequent generation of C5a and C5b-9, the mechanisms and localizationof C5a and C5b-9 during activation are shown be applicant to be toolsfor mediating immune activity. The complement activation occurs viathree main pathways that converge at the C5 convertase step. The C5convertase activity to split the C5 molecule into C5a and C5b isprovided by C3bBb3b, in the alternative pathway convertase and byC4b2aC3b, in the classical pathway convertase. In either case, the C5afragment is released and the two-chain C5b fragment provides thesubstrate for the formation of the C5b-9. The larger fragment C5bassociates with C6 and C7, which forms an amphiphilic entity, capable ofinserting itself into the cell membrane. C8 then joins the complex andunwinds in the membrane. Finally up to sixteen molecules of C9 join toform C5b-9 complex.

It is known that the cleavage of C5 occurs in a fluid phase, thusgenerating C5b and C5a. The association of C5b to C6 and C7 occurs inthe fluid phase leading to the formation of C5b-7 complex, which theninserts itself into the plasma membrane. The fate of soluble C5b-7complex is determined by the soluble complement inactivating factorssuch as S protein, clusterin or vitronectin present in soluble phase.The binding of C5b-7 to these proteins inhibits the insertion of thecomplex into the cell membrane. In a rat alveolar macrophage model itwas reported that soluble MAC, which has little ability to bind to cellsurfaces, did not enhance lung injury after intrapulmonary deposition ofIgG CIC [Czermak et al, Am. J. Pathol. 154(5):1513 (1999),].

Recent work on the C5b-9 molecular complex has shown the importance ofthe C5b-9 molecular complex in the apoptosis, necrosis andpro-inflammatory pathways [Bohana-Kashtan et al, Molecular Immunology41:583 (2004)]. The C5b-9 complex is the principal mediator of injuryinduced by antibodies experimentally directed against glomerular cellmembranes. C5b-9 in sublytic concentration enhances the production ofendothelial intercellular adhesion molecule-1 (ICAM-1) and E selectin,while directly inducing production of interleukin 8 (IL-8) and monocytechemo attractant protein-1 (MCP-1) [Kilgore et al, J. Immunol.155:1434(1995)]. It also has been reported that the C5b-9 complexactivates transcription factors nuclear factor-κ B (NF-κ B) and AP-1resulting in the production of interleukin-6 (IL-6) and interleukin-8 inhuman smooth muscle cells [Viedt et al, FASEB J. 14:2370(2000].

In podocytes, the number of C5b-9 complexes inserted into cell membranesdetermines whether a cell undergoes necrosis. Formation of sublyticC5b-9 complex on the cell membrane results in release of calcium,activation of specific signaling pathways, and an increase in growthfactor production, as well as increased oxidants and proteases [CouserW. G. J. Am. Soc. Nephrol. 1:13(1990); Cybulsky et al, Am J. Pathol.155:1701(1999)]. The sublytic dose of C5b-9 complex on cell membraneactivated cell cycle related genes, i.e. p53, p21, growth arrest DNAdamage-45 (GADD45), checkpoint kinase-1 (CHK-1) and CHK-2. The extracellular signal-regulated kinase (ERK) is involved in a critical pathwayinvolved in regulating these cell cycle related proteins following C5b-9induced DNA damage [Pippin et al, J. Clin. Invest. 111:877 (2003).

Assembly of C5b-9 on cells of the arterial wall induces cell lysis. Thesublytic assembly of C5b-9 on smooth muscle cells and endothelial cellsinduce cell activation and proliferation. Sublytic assembly of C5b-9 onthe plasma membrane activates p 38 MAPK, Janus kinase (JAK) 1, signaltransducer and activator (STAT) 3 and STAT 4 in endothelial cells.[Niculescu et al, J. Immunol. 158:4405 (1997)].

In the passive Heymannn nephritis model of membranous nephropathy, theassembly of C5b-9 induces glomerular epithelial cell (GEC) injury andproteinuria that is partially mediated via production of eicosanoids.The sublytic formation of C5b-9 induces phosphorylation of epidermalgrowth factor receptor (EGF-R), fibroblast growth factor receptor-2 andhepatocyte growth factor receptor. The phosphorylation of tyrosine (204)of ERK-2 as well as free [(3) H] arachidonic acid (AA) and prostaglandinE (2) was stimulated by the formation of C5b-9 on cell membrane. It hasbeen concluded that C5b-9 induces trans-activation of receptor tyrosinekinases in association with ERK2 activation, AA release and PGE (2)production in cultured glomerular epithelial cells (GEC) andglomerulonephritis. [Cybulsky et al, Am. J. Pathol. 155:1701(1999)].

Complement activation and membrane assembly of sublytic C5b-9 play animportant role in inflammation by promoting cell proliferation and byrescuing cell apoptosis. The Sublytic concentrations of C5b-9 increaseCa⁺ influx, activate phospholipases, increase level of diacylglycerol(DAG) & ceramide, activates protein kinase C (PKC) and generatearachidonic acid. In post-mitotic cells such as oligodendrocytes (OLG)and skeletal muscles, C5b-9 reverses the differentiation of the cellphenotype. [Shirazi et al, J. Neurochem. 48:271(1987)]. Sublytic C5b-9also induces proto-oncogenes, activates the cell cycle, and enhancessurvival by inhibiting apoptosis. [Rus et al, J. Immunol.156:4892(1996); Halperin et al, J. Clin. Invest. 91:1974(1993)]. In OLGloss of differentiation due to C5b-9 attack was associated with theactivation of proto-oncogene c-jun, c-fos, and junD and induction of AP1DNA binding activity. C5b-9 is the most potent ERK1 inducer. ERK1activation was preceded by activation of membrane-associated Gi, Ras andRaf-1 then activation of cytoplasmic MAPK/ERK kinase (MEK) 1. TrimericGi protein was also activated [Niculescu et al, J. Immunol.158:4405(1997); Niculescu et al, J. Biol. Chem. 269:4417(1994)].

Complement proteins also play a key role in neurodegenerative diseasessuch as Alzheimer. In several models it has been demonstrated that C5a,via an anti-apoptotic activity provides neuroprotection [Mukherejee etal, J. Neurochem. 77:43 (2001)]. Thus, it is important to specificallyblock the formation of MAC on the CIC thereby regulating the transfer ofMAC in significant doses to avoid the necrosis of the neuronal tissue.The tissue damage by complement and the lytic doses of the MAC leadingto tissue necrosis and inflammatory responses has been the key mechanismin diseases such as lung injury, injury to podocytes, cardiomyopathies,myasthenia gravis, multiple sclerosis, cerebral lupus erythematosus,Guilain-Barre syndrome, Alzheimer's disease, lupus nephritis, membranousnephritis, membrane proliferative glomerulonephritis, rheumatoidarthritis, systemic lupus erythematosus, Behcet's syndrome, juvenileidiopathic arthritis, Sjogren's syndrome, atheroma, thyroiditis,infertility, vasculitis, post bypass syndrome, and tissue incompatibletransplantation.

SUMMARY OF THE INVENTION

The invention relates to the formation of Membrane Attack Complex (MAC)[also referred as Terminal Complement Complex (TCC) or C5b-9] on CIC andthe applications of measuring complement products including C1q, C3, C4,C5, MAC and its components on CIC as diagnostic markers and reduction inthe amount of the MAC on CIC by blocking the formation of MAC on CIC orselectively removing the MAC from the CIC for treatment or prophylaxisof the complement and CIC mediated diseases.

The present invention describes the presence of non-covalentlyassociated complement proteins C1q, C3, C4, C5 and C5b-9 with the CIC.The invention describes for the first time the formation of MAC on theCIC. The experimental data presented in the invention describes that theMAC and components of early complement activation i.e. C1q, C3, C4, C5and C5b-9 present on CIC are not linked to CIC via covalent linkage. Theinvention describes that during plasmapheresis the activated complementproteins C1q, C3, C4, C5 and MAC present on CIC are released from theCIC changing the nature of CIC from pathogenic to non-pathogenic. Theinvention provides methods for measuring the complement activationproducts i.e C1 q, C3, C4, C5 and MAC on CIC and their usefulness inmonitoring the disease activity, and therapy status of complement andCIC mediated diseases including infectious diseases. The invention alsoprovides methods for developing therapies to block the formation of theexcessive MAC on CIC to provide beneficial effects in such diseasesincluding autoimmune disorders, cardiovascular disorders, hematologicaldisorders, oncological disorders, kidney diseases.

BRIEF DESCRIPTION OF DRAWINGS

In the drawings forming the disclosure of this invention:

FIG. 1 illustrates the binding of AHG (aggregated human gamma-globulin)used as immune complex model. A standard curve for binding of AHG wasgenerated in ELISA technique. A linear binding was achieved using theconcentration of 2.34, 4.68, 9.37, 18.75, 37.5, 75, 150 and 300 μg/ml ofAHG. The appropriate dilutions were made in PBS/Tween 20.

FIG. 2 illustrates the presence of CIC with IgG, IgA and IgMimmunoglobulin isotypes in patient's plasma suffering from SLE and RA.

FIG. 3 demonstrate the presence of activated complement components C1q,C3, C4, C5 and C5b-9 in the CIC present bound to CIC in patient plasmafrom autoimmune diseases.

FIG. 4 illustrates the standard curves for (a) C1q, (b) C3, (c) C4, (d)C5 and (e) IgG-CIC used for determining the concentration of therespective components present within the CIC from the patient plasmasample.

FIG. 5 demonstrates the effect of 25 mM EDTA (ethylene diaminetetracetic acid) on the binding of activated complement protein C5 andC5b-9 to CIC. The samples subjected to estimation of C5 and C5b-9 wasmixed with appropriate concentration of EDTA so as to bring the finalconcentration to 25 mM at pH 7.5. Two parallel sets of paired samplesfrom the same patient, one treated with EDTA and other control groupwithout EDTA were subjected to C5 and C5b-9 estimation. As demonstratedin the Figure the treatment of plasma with EDTA decreased the quantitiesof the complement proteins associated with CIC by several fold. It wasconcluded from this experiment that the complement proteins in these CICwere not linked with them due to covalent linkage.

FIG. 6 demonstrates the effect of plasmapheresis on complement bindingto CIC. Sequential samples from patient's undergoing kidney and hearttransplant and treated with plasmapheresis to achieve beneficialclinical results were obtained for analysis. These samples were thenanalyzed for the amount of IgG-CIC, IgA-CIC and IgM-CIC (IgG-CIC, CICcomposed of IgG, IgA-CIC, CIC composed of IgA, IgM-CIC, CIC composed ofIgM). The samples were subjected to measurement of complement proteinsC1q, C3, C4, C5 and C5b-9 bound to CIC pre and post plasmapheresis. Asdemonstrated in FIG. 6, the complement levels bound to CIC decreasedsignificantly with the plasmapheresis. This provided an addedexperimental proof that in vivo interaction of complement with CIC isnot mediated by covalent linkage.

FIG. 7 demonstrates the 2D SDS-PAGE (two dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis) of CIC purified from arheumatoid arthritis patient. The CIC were purified using an affinityresin made by coupling the receptor protein isolated as per patentpublication (PCT/US02/24301). The receptor protein was coupled to NHSactivated Sepharose beads (NHS—Sepharose 4B FF, Pharmacia AB,Piscataway, N.J., US). The resin was packed in a volume of 1.5 ml in asterile disposable polystyrene column. The patient plasma was allowed tointeract with the resin and after washing the non-bound material, theCIC were eluted by lowering the pH to 3.5 with Glycine-HCl buffer. Thepurified CIC were reduced using DTT and 2ME and the proteins were firstseparated based on charge in the first dimension using IEF and based onmolecular weight on the second dimension by polyacrylamide gelelectrophoresis. The CIC components were recognized by comparing the gelimage to 2D SDS-PAGE image of human serum proteins in the NBRF proteindatabase. The CIC purified with the affinity column displayed thepresence of heavy chain of globulins both γ and μ heavy chains and bothlight K and λ light chains thereby confirming the identity of CIC. Theassociation of acute phase serum protein CRP was also identifiedassociated in the CIC.

DETAILED DESCRIPTION

The following terms have the following meaning:Circulating Immune Complexes (CIC): Antigen antibody complexes thatcirculation in plasma are referred as circulating immune complexes.Apoptosis: Programmed cell death. A process of cell death characterizedby DNA cleavage, nuclear condensation, and plasma membrane blebbing thatleads to phagocytosis of cell without undergoing inflammation.Necrosis: The sum of morphological changes indicative of cell death andcaused by progressive degradative action of enzymes, associated withinflammation.Innate immunity: Protection against infection that relies on mechanismsthat exist before infection, are capable of rapid response to microbesand reacts in essentially same way to repeated infections.Adoptive immunity: The form of immunity that is mediated by lymphocytesand stimulated by exposure to infectious agent.Mimotope: An entity that can mimic the antigenic epitope.Pleitropic: Triggering of multiple function by the same molecule.

As used herein, the term “isolated” means captured in a manner whichrenders the composition useful for one or more of the diagnostic ortherapeutic purposes described herein. Using receptors isolated(prepared as detailed in PCT 20, 010, 801 Chauhan et al, incorporated byreference herein) from cell lines, applicant for the first time hasdemonstrated the presence of C5 and MAC on CIC. The second importantfinding reported in the instant application is that complement bound tothe CIC is not linked by covalent bond. Thus CIC acts as a substrate forformation of C5B-9 and subsequent transfer to the target cell surface.The presence of higher amounts of C5b-9 on CIC leads to the transfer oflytic doses of C5B-9 to cell surface leading to necrosis, the sublyticdoses of C5b-9 lead to cell activation, proliferation, apoptosis and anumber of other cellular events. Biochemical or biological moleculesrestricting the formation of MAC on CIC provides a therapeutic target asthe reducing the amount of MAC from lytic doses to sublytic is abeneficial approach for treating complement and CIC mediated injuries.

In the experiments, receptors were isolated from a lymphoblastoid cellline that binds to CIC composed with IgG, IgM and IgA isotypes ofimmunoglobulin. These complexes are composed of antigen, antibody andother acute phase reactants from the patient plasma such as complementproteins, C reactive protein and serum amyloid protein (SAP).Subsequently, after capturing the CIC on a solid phase coated with thereceptor, the CIC was analyzed for their composition. In order toanalyze the composition of the CIC from disease patients, ELISA basedassays were used. These assays were developed by coating the ELISAplates with the receptor specific for capture of the CIC. The capturedCIC on the ELISA plates were probed with secondary antibodies in a twostep process with enzyme coupled to antibodies. To demonstrate thepresence of complement proteins in the CIC, antibodies directed tocomplement proteins were specifically used to demonstrate their presencein the CIC. No external additive was added to the interaction, thus nooutside interference from other proteins was involved. A previouslytitrated secondary antibody-HRP conjugate was used to measure thebinding of isotype specific CIC. For measuring the complement proteinsbound to CIC, the antiserum specific to each complement protein wasallowed to interact with the complex bound to the solid phase of theELISA plates. Subsequently, secondary antibody-HRP conjugate directed tothis serum was used for measuring the amount of complement proteinsbound within these complexes. Purified proteins and in vitro formedcomplexes were used as standards in separate wells to quantitateconstituents of these CIC.

In our analysis, serum samples from patients with rheumatoid arthritis(RA) and systemic lupus erythematosus (SLE) were used to demonstratethat during disease activity, the CIC evidence significant variation intheir composition with respect to the presence of immunoglobulinisotypes IgG, IgA and IgM. In addition to the composition of the CICisotypes, the sera from these patients also demonstrated significantlevels of complement proteins C1q, C3 C4, C5 and C5b-9 associated withthe CIC. Opsonization of CIC with these complement proteins can clearapoptotic debris generated during the normal physiology, as well asduring an infection. Any defect in clearance of CIC, or the excessiveformation of CIC during infection is a major pathologic event resultingin development of auto immune disorders [Walport M J, Arthritis Res.4(suppl 3):S279 (2002)]. For the first time, however, applicant hasdemonstrated the presence of the C5 component of complement and thereactivity to C5b-9 bound to the CIC. However using the antibodiesdirected towards C5a, no reactivity was detected, thus suggesting thatC5a, after cleavage from the C5b, falls into the soluble phase and doesnot form a part of the complex.

Thus, split product of complement C3b and C4b binds to proteins via theformation of a thiol ester bond that gets exposed via activation. Theproduct of the C4A gene binds to CIC by formation of an amide bond,while the product of the C4B gene binds via a carboxyl group throughesterification. For the first time applicant demonstrates that the majorportion of the activated complement components present on CIC are notcovalently attached but only a small portion of the complement appearsto be linked via covalent linkage. In patients undergoingplasmapheresis, it was observed by us that post therapy the levels ofthe complement proteins C1q, C3, C4, C5 and MAC associated with CIC weresignificantly reduced. It is postulated that the beneficial effect ofthe plasmapheresis therapy in these patients is mediated due to decreasein the complement proteins associated with the CIC. To confirm this factwe performed two experiments. In one experiment, the effect of EDTA onthe complement proteins bound to CIC was analyzed. In this experiment wedemonstrated that by treating the serum sample with ethylene diaminetetra acetic acid (EDTA), complement proteins bound to the CIC arereleased. Serum samples that had been previously tested for the presenceof CIC and complement in the presence and absence of EDTA were analyzed.The serum samples from five patients were diluted 1:20 with PBScontaining 0.05% Tween 20. One set of samples was treated with 25 mMEDTA that was included in the diluent buffer. The inclusion of EDTAdramatically reduced the binding of complement proteins C3, C4, C5 andC5b-9 in the CIC. Thus, the inclusion of 25 mM EDTA in the PBS as adiluent, and subsequently in the incubation step during the binding,while not affecting the amount of immunoglobulin isotype amounts,eliminated the presence of the complement proteins associated with theCIC. The covalent linkage of the complement to CIC tags these moleculesto be cleared by binding with the complement receptor 1 (CR 1) presenton the erythrocytes and therefore makes them biologically non-availablefor signaling functions attributed to the complement. Since thenon-covalently associated complement is biologically available it can beutilized for signaling pathways and thus can play key role in thedisease pathogenesis.

In another experiment the patient serum samples collected from the preand post plasmapheresis were analyzed for the presence of complementproteins bound to CIC. In plasmapheresis therapy the one fifth of theplasma volume from patient was replaced with the 5% human serum albuminusing the extracorporeal circuit. The experiment demonstrated thatplasmapheresis affects the binding of complement proteins to CIC. Adramatic drop from 40 to 90% was demonstrated for various complementproteins which bind to the CIC. Accordingly, the beneficial effect ofthe therapy was provided.

The nature of the CIC of the samples was established by selectivelyisolating CIC from patient with RA and SLE. The CIC was purified usingan affinity resin developed by coupling the receptor preparation withSepharose FF 4B (Pharmacia, Piscataway N.J.). The affinity resin wasthen utilized to capture CIC from the RA and SLE patients. Theindividual components of CIC were displayed on SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) and the individualcomponents were recognized by Western Blotting. The identity ofindividual immunoglobulin chains was established using μ and γ heavychain specific antibodies. The identity of the components of the CIC wasalso established by subjecting the CIC to 2D SDS-PAGE analysis. Usingthe comparative analysis of the results from the NBRF protein database,the presence of IgM and IgG heavy chains as well as both kappa andlambda light chains in the captured CIC was established.

The measurement of CIC, composed with various immunoglobulin isotypesand the activated complement component C1q, C2, C3, C4, C5, and C5b-9bound to CIC, provides a useful indicator as one aspect of theinvention. In this aspect, the modulation of the complement on CIC by anappropriate chemical, biochemical, peptide or biological is useful inthe treatment of disease, including: Renal Diseases Anti-GlomerularBasement Membrane Disease Renal Vasculitis: Focal NecrotizingGlomerulonephritisoRapidly progressive glomerulonephritisoWegener”sgranulomatosus (WG)oMicroscopic polyangitisoldiopathic RPGN FocalSegmental Glomerulosclerosis Systemic Lupus ErythematosusAnti-Glomerular Basement Membrane Disease Neurological DiseaseEaton-Lambert Syndrome Guillain-Barre” syndrome Amyotrophic LateralSclerosis Myasthenia Gravis Inflammatory Polyneuropathy MultipleSclerosis Alzheimer Hematological Disease Mycelia and CryoglobulinemiaThrombotic Thrombocytopenic Purpura Idiopathic Thrombocytopenic PurpuraAllaoantibodies in Hematologic Disease Rheumatologic Disease RheumatoidArthritis Rheumatoid Vasculitis Scleroderma Dermator Early stages ofScleroderma Dermatomyosistis Polymyosistis Sjogren”s syndrome Behcet”sdisease Other Disease Pemphigus Vulguris associated to antibodies tosquamous epithelium Bullous pemphigoid associated to antibodies todermal basement Cardiovascular Disease: Myocardial InfarctionCardiomyopathies Ischemia reperfusion injury Transplant NeoplasticDiseases

EXAMPLES Example One

The example describes the development and use of ELISA based assays formeasurement of components of CIC i.e. Antibody isotypes, IgG (IgG1,IgG2, IgG3, IgG4), IgA, IgM; complement proteins, C1q, C3, C4, C5 andC5b-9; other acute phase proteins associated within the CIC.

Details on performing the assay for measuring the components of CIC: (1)Purified receptor preparation the proteins binding specifically to CICvia Fc portion were dissolved in an alkaline buffer (0.1M sodiumcarbonate pH 9.6).

(2) The alkaline solution with receptor preparation was placed incontact with the plate at 4° C. for 12 to 24 hours.

(3) The coating was removed form the plate and plate washed three timeswith a solution of sodium chloride (0.15M), buffered by sodium andpotassium phosphate (0.01M, pH 7.2 to 7.4) (PBS) to remove unboundreceptors.

(4) Thereafter to block free sites the plate was placed in contact with100 μl of 1% BSA dissolved in PBS containing 0.05% Tween-20.

(5) The blocking solution was removed and plates washed three times withPBS fortified with 0.05% of Tween-20 (v/v).

(6) The sera from patients were diluted properly with a solution of PBSprior to testing. In this example we diluted the sera 10 volumes and 20volumes of PBS.

(7) A total of 0.1 ml aliquots of diluted sera were placed intoappropriately designated wells. For this example duplicatedeterminations were performed for each specimen and average values wereused for calculations.

(8) The plates were kept at 37° C. in humid container for two hours.

(9) The plates were washed again with PBS/Tween-20.

(10) The plates were filled with 100 μl of appropriate anti serum formeasurements (anti-human IgG-HRP, anti-human IgM-HRP, anti-humanIgA-HRP, anti-human C3, anti-human C4, anti-human C1q, anti-human C5 andanti-human C5b-9). The plates were incubated at room temperature forsixty minutes.

(11) The plates were washed again and the plates that received the HRPconjugates were developed for HRP enzyme activity. Otherwise the wellsin plates without conjugated antiserum were filled species, specificanti-HRP conjugate and further incubated for sixty minutes at roomtemperature.

(12) After washing each plate well in the plate was assayed forhorseradish peroxidase activity by addition of 100 μl substrate buffer(TMB substrate).

(13) The reaction was monitored for the development of color and atappropriate color density the reaction was terminated by addition of 25μl of 2.5 M H₂SO₄.

(14) Optical Density was measured and plotted against the standardsconcentrations and the linear equation was used to obtain the quantityof components in the CIC (FIG. 4).

Example Two Purification and Analysis of CIC on 2D SDS-PAGE:

(1) The receptor protein binding was conjugated to NHS-activated(n-hydroxyl succinamide) sepharose 4 B (Pharmacia, Piscataway, USA).

(2) The free sites on the resin were blocked with excess of 1M Tris-HClpH 7.5.

(3) The resin was washed with PBS to remove unbound Tris-HCl.

(4) In a column with one ml of resin a total of 1.5 ml of patient plasmawas placed in contact with the receptor bound resin.

(5) The plasma was allowed to flow under gravity and fifteen times PBSwas allowed to flow into the column to remove unbound plasma proteins.

(6) The bound CIC were eluted with low pH buffer (Glycine-HCl, 0.1M, pH3.5).

(7) Captured CIC were concentrated to a final volume of 300 μl.

(8) Twenty micro-liters of the purified CIC proteins were mixed with IEFrenaturing solution consisting of 8M Urea, Bridge 58, NP40, 2ME,β-Octylglucoside.

(9) The sample was mixed properly and applied to 7 mm IPG strips(Bio-Rad, Hercules, Calif.). The strips were left for 16 hours at roomtemperature thereafter the IPG strips were subjected to isoelectricfocusing on pH 3.5 to 10 IPG strip in accordance with manufacturerrecommendation.

(10) A total amount of 10,000 Volt-hours were applied during the IEF.

(11) After the isoelectric focusing the strips were removed from the IEFcell and drained of excess mineral oil and incubated with buffercontaining 8M Urea, 0.375 M of Tris-HCl buffer pH 8.8, 20% Glycerol, 100mM DTT for 15 minute with constant shaking.

(12) After the first incubation the IEF strips were incubated foranother fifteen minute at room temperature in a buffer with compositionsimilar to earlier buffer containing 125 mg of Iodoacteamide per 10 mlof buffer.

(13) Thereafter the IPG strips were overlaid for second dimension run on4 to 12% SDS-PAGE NuPAGE gel (Invitrogen, Carlsbad, Calif.).

(14) The electrophoresis was carried out in MOPS buffer at 170 volts fortwo hours. The gels were then fixed in acetic acid and ethanol fixative.

(15) The gels were stained with silver stain. The comparative analysisof the, CIC were done utilizing the 2D protein database from EMBO toestablish the identity of globulin heavy and light chains.

It is to be understood that the present invention has been described indetail by way of illustration and example in order to acquaint othersskilled in the art with the invention, its principles, and its practicalapplication. Further, the specific embodiments of the present inventionas set forth are not intended as being exhaustive or limiting of theinvention, and that many alternatives, modifications, and variationswill be apparent to those skilled in the art in light of the foregoingexamples and detailed description. Accordingly, this invention isintended to embrace all such alternatives, modifications, and variationsthat fall within the spirit and scope of the following claims. Whilesome of the examples and descriptions above include some conclusionsabout the way the invention may function, the inventor does not intendto be bound by those conclusions and functions, but puts them forth onlyas possible explanations.

All references cited herein are hereby incorporated by reference asthough fully set forth in the application

1. An isolated complex comprising one or both of complement activationproduct C5, and membrane attack complex (C5b-9) associated withcirculating immune complex.
 2. A method for inhibiting the formation ofa non-covalent combination of membrane attack complex and circulatingimmune complex comprising application of an inhibitor selected from thegroup consisting of a monoclonal antibody, peptide mimotope, or smallmolecule in patients suffering from complement and circulating immunecomplex mediated diseases.
 3. A method for screening candidatecompositions or processes for an ability for inhibiting the formation ofmembrane attack complex on circulating immune complex comprisingassessing the composition or process for the reduction in membraneattack complex associated with circulating immune complex as a result ofthe application of the candidate composition or process.
 4. A method ofmonitoring the formation of membrane attack complex and other splitproducts of C5 on circulating immune complex from the serum, plasma,cerebrospinal fluid and other bodily fluids in diseases associated withcomplement and circulating immune complex pathogenesis comprisingmeasuring the formation of said products and assessing for symptoms ofsaid disease.
 5. Isolated complexes comprising one or more of the groupconsisting of non-covalent linked complement split products C1q, C3, C4,C5 and membrane attack complex on circulating immune complex.
 6. Amethod of inhibiting non-covalent association of C1q, C3, C4, C5 andmembrane attack complex to circulating immune complex comprisingapplication of an inhibitor selected from the group consisting of amonoclonal antibody, peptide mimotope, or small molecule in patientssuffering from complement and circulating immune complex mediateddiseases. 7-8. (canceled)
 9. A process for quantitative measurement forthe presence of complement C5 and C5b-9 associated with circulatingimmune complex, the process comprising the following steps: a. Providinga test device comprising a receptor preparation in solid phase as acapture reagent for circulating immune complex; b. Establishing aselected working range for an immunoassay within said ranges ofcomposition of circulating immune complex, IgG-CIC 2 to 1000 μg/ml,IgA-CIC 0 to 1000 μg/ml, IgM-CIC 0 to μg/ml, C1q bound to CIC 0 to 10μg/ml, C3 bound to CIC 0 to 30 μg/ml, C4 bound to CIC 0 to 10 μg/ml, C5bound to CIC 0 to 10 μg/ml and C5b-9 0 to 10 μg/ml; c. Constructing astandard assay curve by plotting relative degree of immunochemicalbinding of said circulating immune complex components to the testdevice; d. Interacting a fixed concentration of immunospecific conjugateof said substances, the composition of complexes resulting from saidimmunological substances and immunospecific conjugate being within theselected working range limits; e. Providing a test system comprising ofsaid test device, said immunospecific conjugate, said immunologicalsubstances, the amount of said immunospecific conjugate beingsubstantially equivalent to said fixed concentration of immunospecificconjugate, and the amount of said immunospecifically determinablesubstance being appropriate to produce a known degree of immunochemicalbinding corresponding to a predetermined point on said standard curve,thereby enabling quantitative assaying of one or more of complementproteins C1q, C3, C4, C5 and C5b-9 present on circulating immunecomplex.
 10. (canceled)
 11. A process for measurement of one or morecomplement proteins C1q, C3, C4, C5 and C5b-9 from plasma or otherbodily fluids of animals suffering from or at risk of suffering from adisease or condition, including but not limited to autoimmune,cardiovascular, neurodegenerative disorders, oncological diseases andinfectious disease, said process comprising: a. Providing a test devicecomprising a receptor preparation in solid phase; b. Establishingselected working ranges for said immunoassay within said ranges forcomplement proteins; c. Constructing a standard assay curve by plottingrelative degree of immunochemical binding of said complementcomponent(s) to the test device; d. Interacting a fixed concentration ofa immunospecific conjugate directed to complement proteins andimmunospecific conjugate being within pre selected working range limits;e. Providing a test system comprising of said test device, saidimmunospecific conjugate, said immunological substances, the amount ofsaid immunospecific conjugate being substantially equivalent to saidfixed concentration of immunospecific conjugate, and the amount of saidimmunospecifically determinable substance being appropriate to produce aknown degree of immunochemical binding corresponding to a predeterminedpoint on said standard curve, thereby enabling quantitative assaying ofone or more of complement C1q, C3, C4, C5 and C5b-9 present oncirculating immune complex.
 12. A process for quantitation ofimmunoglobulin isotype composition of circulating immune complex orantigens bound within circulating immune complex comprising using anELISA based on receptor based capture mechanism, said processcomprising: a. Placing the receptor on solid phase of ELISA plates,micro beads or other suitable surface; b. Attaching the biotin or otherform of detection tag on the antigen or antibody; c. Mixing the taggedantigen or antibody with the patient plasma, patient serum, sinuovialfluid, cerebrospinal fluid (CSF) or other bodily fluid; d. Placing themixture in contact with receptor attached to the solid surface; e.Washing the unbound components with buffers; f. Quantitating the taggedantigen or antibody with a reagent including, but not limited toAvidin-Horse Radish Peroxidase and color development reagents.
 13. Aprocess as set forth in claim 3 for screening the composition of ablocking agent for the formation of membrane attack complex anddeposition of C5 on circulating immune complex.
 14. (canceled)
 15. Aprocess for screening a composition that targets blocking of complementactivation or other component assembly in the circulating immune complexas set forth in claims 11, and modulating the binding of serum acutephase proteins bound to circulating immune complex, said processcomprising: a. Attaching the receptor to solid phase or studying theinteraction in the liquid phase, allowing the interaction of thecirculating immune complex with the receptor in presence of complementproteins to activate complement deposition or other acute phase proteinson circulating immune complex; b. Placing the blocking compositionduring the activation of the complement on circulating immune complex orassociation of serum acute phase protein; c. The composition beingselected from the group consisting of a chemical, biochemical, protein,peptide and monoclonal; d. Obtaining initial data indicating whether theformation of membrane attack complex and binding of complement C1q, C2,C3, C4, and C5 is inhibited on the circulating immune complex; e.Obtaining data indicating whether the serum acute phase proteinsassociated with the circulating immune complex is inhibited. 16-17.(canceled)
 18. A method of reducing disease symptoms in an individualcomprising: identifying an individual in need of reducing the symptomsdue to increased complement fixation on circulating immune complexleading to inflammation and tissue necrosis by administering acomposition comprising a monoclonal antibody, peptide, mimotope oractive molecule
 19. A process in accordance with claim 11 that furthercomprises contacting a receptor during interaction with circulatingimmune complex and complement with at least one of a humanizedmonoclonal antibodies, active molecules, peptides and mimotopes andobtaining data indicative of whether the activation of complement hasbeen inhibited.
 20. A process in accordance with claim 17 that furthercomprises inoculating patients or animals with the immune complex andcomposition, wherein the immune complex mediated immune responses arealtered providing beneficial effect.
 21. The method of claim 2 whereinthe disease is selected from the group consisting of systemic lupuserythematosus, rheumatoid arthritis, cardiovascular diseases, kidneydiseases, and autoimmune diseases.
 22. The method of claim 4 wherein thedisease is selected from the group consisting of autoimmune diseases,cardiovascular diseases, neurodegenerative diseases, infectious diseaseand oncological diseases.
 23. The method of claim 6 wherein the diseaseis selected from the group consisting of systemic lupus erythematosus,rheumatoid arthritis, cardiovascular diseases, kidney diseases, andautoimmune diseases.